Chondroitinase ABC I and methods of analyzing therewith

Abstract

The invention relates to chondroitinase ABC I and uses thereof. In particular, the invention relates to recombinant and modified chondroitinase ABC I, their production and their uses. The chondroitinase ABC I enzymes of the invention are useful for a variety of purposes, including degrading and analyzing polysaccharides such as glycosaminoglycans (GAGs). These GAGs can include chondroitin sulfate, dermatan sulfate, unsulfated chondroitin and hyaluronan. The chondroitinase ABC I enzymes can also be used in therapeutic methods such as promoting nerve regeneration, promoting stroke recovery, treating spinal cord injury, treating epithelial disease, treating infections and treating cancer.

We claim: 
     
       1. A method of analyzing a sample of polysaccharides, comprising: contacting the sample with a modified chondroitinase ABC I in an amount effective to degrade the polysaccharides, wherein the modified chondroitinase ABC I comprises the amino acid sequence of the native chondroitinase ABC I encoded by the nucleic acid sequence of SEQ ID NO: 1, wherein at least one substrate specific residue has been substituted with a different amino acid than in the native chondroitinase ABC I, and wherein the nucleic acid that encodes the modified chondroitinase ABC I is at least 95% homologous to the nucleic acid of SEQ ID NO:1, and wherein the amino acid sequence of the modified chondroitinase ABC I is not the amino acid sequence of any of SEQ ID NOs: 3-24, wherein the at least one substrate specific residue is the residue that corresponds to the residue at position 105, 221, 312, 388, 389, 392, 395, 500, 501, 508, 560 or 653 of the native chondroitinase ABC I of SEQ ID NO: 2. 
     
     
       2. The method of  claim 1 , wherein the residue at position 221 has been substituted with alanine, lysine, methionine or glutamine. 
     
     
       3. The method of  claim 1 , wherein the residue at position 312 has been substituted with alanine. 
     
     
       4. The method of  claim 1 , wherein the residue at position 388 has been substituted with alanine, lysine or arginine. 
     
     
       5. The method of  claim 1 , wherein the residue at position 389 has been substituted with alanine, lysine or arginine. 
     
     
       6. The method of  claim 1 , wherein the residue at position 392 has been substituted with alanine or phenylalanine. 
     
     
       7. The method of  claim 1 , wherein the residue at position 500 has been substituted with alanine, cysteine or glutamine. 
     
     
       8. The method of  claim 1 , wherein the residue at position 501 has been substituted with alanine, lysine or arginine. 
     
     
       9. The method of  claim 1 , wherein the residue at position 508 has been substituted with phenylalanine. 
     
     
       10. The method of  claim 1 , wherein the residue at position 560 has been substituted with alanine or lysine. 
     
     
       11. The method of  claim 1 , wherein the residue at position 653 has been substituted with alanine, aspartic acid or glutamine. 
     
     
       12. The method of  claim 1 , wherein the sample is a sample of glycosaminoglycan. 
     
     
       13. The method of  claim 1 , wherein the sample is a sample of galactosaminoglycan. 
     
     
       14. The method of  claim 1 , wherein the substituted amino acid is a conservative amino acid substitution. 
     
     
       15. The method of  claim 1 , wherein the modified chondroitinase ABC I is a substantially purified recombinant form. 
     
     
       16. The method of  claim 1 , wherein the modified chondroitinase ABC I has a k cat  or K m  value for a substrate that is at least 10% different than a native chondroitinase ABC I k cat  or K m  value. 
     
     
       17. The method of  claim 16 , wherein the modified chondroitinase ABC I k cat  or K m  value is at least 20% different than a native chondroitinase ABC I k cat  or K m  value. 
     
     
       18. The method of  claim 17 , wherein the modified chondroitinase ABC I k cat  or K m  value is at least 50% different than a native chondroitinase ABC I k cat  or K m  value. 
     
     
       19. The method of  claim 16 , wherein the substrate is a glycosaminoglycan. 
     
     
       20. The method of  claim 16 , wherein the substrate is a galactosaminoglycan. 
     
     
       21. The method of  claim 1 , wherein the nucleic acid that encodes the modified chondroitinase ABC I is at least 97% homologous to the nucleic acid of the native chondroitinase ABC I. 
     
     
       22. The method of  claim 21 , wherein the nucleic acid that encodes the modified chondroitinase ABC I is at least 99% homologous to the nucleic acid of the native chondroitinase ABC I. 
     
     
       23. The method of  claim 1 , wherein the modified chondroitinase ABC I selectively degrades chondroitin sulfate or dermatan sulfate. 
     
     
       24. The method of  claim 23 , wherein the residue at position 388 has been substituted with alanine, lysine or arginine. 
     
     
       25. The method of  claim 23 , wherein the residue at position 389 has been substituted with alanine, lysine or arginine. 
     
     
       26. The method of  claim 23 , wherein the residue at position 500 has been substituted with alanine. 
     
     
       27. The method of  claim 23 , wherein the residue at position 653 has been substituted with alanine, lysine or arginine. 
     
     
       28. The method of  claim 1 , wherein the modified chondroitinase ABC I selectively degrades dermatan sulfate. 
     
     
       29. The method of  claim 28 , wherein the residue at position 560 has been substituted with alanine or lysine. 
     
     
       30. The method of  claim 1 , wherein the modified chondroitinase ABC I selectively degrades chondroitin 6-sulfate or chondroitin 4-sulfate. 
     
     
       31. The method of  claim 30 , wherein the residue at position 500 has been substituted with cysteine or lysine. 
     
     
       32. The method of  claim 1 , wherein the modified chondroitinase ABC I selectively degrades chondroitin 4-sulfate. 
     
     
       33. The method of  claim 32 , wherein the residue at position 221 has been substituted with alanine. 
     
     
       34. The method of  claim 32 , wherein the residue at position 500 has been substituted with glutamine.